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Can P. putida :SD denitrify?

We integrated the complete denitrification machinery in P. putida and tested whether the denitrification BAC worked by measuring nitrate reduction, nitrite accumulation, nitrous oxide accumulation. Nitrate reduction was measured to test the overall efficiency of the pathway. However, this assay was not sensitive enough to conclude whether the pathway was active.

Is Nap active in for P. putida :SD

To test Nap activity for P. putida :SD we performed a Griess assay to quantify nitrite accumulation (Figure 1). Additionally, we tested the effect of IPTG induction on the accumulation of nitrite. For this condition, :SD+, the denitrification pathway is expressed to a higher extend. With IPTG, overall expression of the system was higher, which was also reflected in the OD₆₀₀. The higher metabolic burden resulted in a OD₆₀₀ 66% lower than that for :SD without IPTG.

Compared to the control P. putida ΔnasT, more nitrite accumulates for both :SD and :SD+, demonstrating that the integrated Nap works. However, when compared with the Nap plasmid, less nitrite accumulates. This could imply that (1) the nitrate reduction was not as optimal as for the Nap plasmid or (2) that Nir was active. Nir reduces nitrite to nitric oxide, this concatenated reduction step could explain the lower amounts of nitrite accumulated for :SD. The fact that :SD+, accumulated less nitrite compared to :SD could be explained by the higher expression of all denitrification genes increasing pathway efficiency.

Figure 1. caption text

Does nitrous oxide accumulate?

Given that we showed that Nap, being first enzyme in the pathway worked in P. putida :SD, we further tested nitrous oxide accumulation. Nitrous oxide accumulation would be the concatenated effect of Nap, Nir and Nor, and measuring its production verifies the activity of these enzymes. Within the timeframe of iGEM, we were able to perform one GC-MS experiment for which we measured the N₂O concentration at t= 0, t= 24, t= 48 and t= 139 in the headspace. Over the timeframe, no N₂O accumulated for P. putida :SD. However, for two out of three biological replicates of P. putida :SD+ we noticed a slight increase in N₂O (Figure 12). These preliminary results suggest that the combined Nap, Nir, Nor enzymes work as nitrous oxide is accumulating slightly. This accumulation could happen due to a lack of electrons, as illustrated by Pan et al. [2]. This could also be explained by the fact that Nos is active in this strain and that it further reduces nitrous oxide, explaining only the small accumulation. However, to know for sure, this experiment needs to be repeated.

Figure 2. caption text

Can strain P. putida :SD reduce nitrous oxide?

Thus far, heterologous expression of Nos in vivo has not been successful. Therefore, to test Nos activity separately, we expressed the Nos accessory plasmidcontaining multiple putative electron donors for this enzyme. Nos activity was tested by culturing P. putida :SD with 0.5% (5000 ppm) N₂O in the headspace and subsequently measuring the reduction in N2O with GC-MS at t=0, t=24, t=48. Unfortunately, no significant reduction of N₂O consumption was observed. We used P. stutzeri JM300 as a positive control to check if this native denitrifier could convert N₂O into N₂, but also no reduction of N₂ was observed. This could indicate that the experimental conditions were not optimal for Nos activity, or that the reduction was so little that it could not be measured with the current GC-MS settings. Similar to the <a class="text-link Mosaic approach, within the time frame of iGEM it was not possible to repeat this experiment.